The task of safeguarding genomic stability is not accomplished by any single cellular process. Instead, it relies on the integrated action of a number of processes including cell cycle progression, DNA replication, DNA repair, and others. The checkpoint signaling pathway initiated by the ATR kinase is a central coordinator and regulator of these processes. Compromised ATR checkpoint is found in cancers and it results in increased sensitivity to DNA damage. Many cancer drugs in clinical use are DMA-damaging agents that activate the ATR checkpoint. The long-term goal of the proposed research is to understand how ATR is regulated by DNA damage. To understand how ATR recognizes DNA damage and how it is activated by DNA damage, we have systematically established biochemical assays to characterize the critical events that lead to ATR activation. We have discovered that ATRIP, the regulatory partner of ATR, associates directly with RPA-coated single-stranded DNA (RPA-ssDNA) and enables the ATR-ATRIP complex to recognize this DNA damage-induced structure. This finding has led us to hypothesize that the ATR checkpoint is activated and regulated by the specific DNA-protein structures induced by DNA damage. Using complex DNA structures with ssDNA regions, we have successfully recapitulated the activation of ATR in vitro. This has presented to us a unique opportunity to identify the DNA and protein structures that activate ATR, and to reveal the mechanisms of ATR activation. Our working hypothesis in this proposal is that ATR is regulated at 3 levels (damage recognition, kinase activation, and substrate recognition) by specific DNA-protein structures at sites of DNA damage. In support of this, we found that (1) ATRIP associates with RPA-ssDNA in an ATR-regulated manner;(2) the kinase activity of ATR can be stimulated by specific DNA structures in vitro;and (3) Rad17 regulates the function of ATR after its activation. Our goal in this proposal is to determine how ATR is regulated at these 3 levels by exploiting the biochemical systems that we developed. Moreover, we will investigate whether inhibition of specific checkpoint responses can benefit cancer therapy. Our specific aims are: (1) Characterize the function and regulation of the ATRIP-RPA-ssDNA interactions. (2) Determine the basic elements and mechanisms of ATR activation. (3) Investigate the functions of Rad17 and Claspin in stress-specific ATR signaling.